PPG phosphoramidite is often used in synthesis of nucleotides and oligonucleotides containing pyrazolopyrimidine base(s). These nucleotides and oligonucleotides are useful in a variety of applications, including as crosslinkable probes for use in therapeutic and diagnostic applications. See for example PCT publication No. WO 90/14353, which is incorporated herein by reference in its entirety. In addition, oligonucleotides in which one or more purine residues have been replaced by pyrazolopyrimidines display enhanced duplex and triplex-forming ability and display enhanced mismatch discrimination ability. See, for example, Belousov et al., Nucleic Acids Res., 1998, 26, 1324-1328 and U.S. Pat. Nos. 5,594,121 and 6,127,121, all of which are incorporated herein by reference in their entirety. Furthermore, PPG containing oligonucleotides have been shown to reduce self-association observed with guanine-rich oligonucleotides and quenching observed when guanine is adjacent to some fluorophore-containing oligonucleotide conjugates. See WO 00/142505.
Conventional syntheses of PPG phosphoramidite involve coupling an activated sugar moiety with a pyrazolopyrimidine base to produce a nucleoside containing pyrazolopyrimidine base. See, for example, Seela et al., Helv. Chim. Acta, 1986, 69, 1602-1613. Further reactions are then carried out to produce PPG phosphoramidite. Unfortunately, conventional processes for producing PPG phosphoramidite require a numerous chromatography separations including that of the nucleoside produced from coupling an activated sugar moiety to a pyrazolopyrimidine base. Typically, chromatography purification of a compound is not suitable for a large scale (e.g., tens or hundreds of grams or more) production because a chromatography process is generally time consuming and labor intensive. Moreover, a large scale chromatography purification process requires a correspondingly large amount of chromatography material, e.g., silica gel and chromatography solvent(s), which increases the production cost of PPG phosphoramidite. Because the pyrazolopyrimidine base containing nucleoside is produced relatively early in the conventional synthesis of PPG phosphoramidite, purification of this nucleoside by chromatography is one of the major hindrances in a cost effective large scale PPG phosphoramidite synthesis.
Therefore, there is a need for a process which is amenable for a large scale production of PPG phosphoramidite.